Urinary incontinence affects more than 200 million patients worldwide, and is associated with significant reduction in quality of life Current treatment options have limitations and options are needed. Cell therapy for urinary sphincter deficiency (USD) - associated stress incontinence in women continues to be tested in preclinical studies and in clinical trials. This is a renewal application fo an RO1 whose initial goals were to develop a nonhuman primate (NHP) model of stable USD and then measure the long-term effects of autologous skeletal muscle precursor cell (skMPC) therapy on sphincter structure and function. Results indicate that transectin the pudendal innervation to the urinary sphincter complex of female NHPs produced stable (up to 12 months after injury) USD and that sphincteric injection of skMPCs produced equally stable structural and functional improvement. The renewal application will utilize the NHP model of USD to address remaining and additional gaps in knowledge concerning this therapeutic approach. 1) Initial studies provide evidence that, at least in part, injected cels actively incorporate into the sphincter tissue~ but that they may also modulate native cell migration to the sphincter. Thus, proposed studies will more definitively explore cell incorporation vs. stimulation of cell migration as central modes of action by which injected cells regenerate sphincter tissue. 2) The efficacy of cell therapy in established/stabl USD - the usual clinical picture - remains unclear and will now be tested in this model. ) Studies will compare the efficacy of direct sphincter vs. intravenous injection - which could afford wider clinical acceptance. Following bone marrow transplantation with labeled autologous labeled bone marrow progenitor cells (BMCs), autologous labeled skMPCs will be administered either 6 weeks post-sphincter denervation, or 6 months post-denervation, either intravenously, or directly into the sphincter complex. Aim 1. To measure and compare the route of skMPC treatment and the timing of skMPC treatment on structural changes of the urinary sphincter complex in the NHP model of USD. The hypothesis is that stimulation of native cell migration is an important process by whih injected cells contribute to regeneration of the sphincter complex. As such, structural regeneration of the sphincter will be greater in the local cell therapy groups (regardless of timing) because initial direct cell-cell content enhances chemotactic paracrine activity and increases migration of native cells (BMCs) to the sphincter compared to systemic injection. Aim 2 is to compare the route and timing of skMPC treatment on functional changes in the urinary sphincter (somatic and adrenergic reflex control of sphincter pressure and blood flow). The hypothesis is that functional regeneration will reflect treatment effects on te structural, vascular and muscular components of sphincter complex. It is anticipated that results of proposed studies will help better define the cell treatment window of opportunity, possible effective routes of administration and mode of action of cell therapy in an anial model that sits at the nexus of clinical translation.